The present invention relates to novel isocoumarin derivatives inhibiting angiogenesis, a method for preparation thereof and pharmaceutical compositions comprising the said derivatives as pharmaceutically active ingredients.
More particularly, the present invention relates to novel isocoumarin derivatives represented by formula 1, especially 6,8-dihydroxy-4-acetyl-isocoumarin, a method for preparing 6,8-dihydroxy-4-acetyl-isocoumarin from fungi, and pharmaceutical compositions comprising the compounds and/or 6,8-dihydroxy-4-acetyl-isocoumarin as pharmaceutically active ingredients, which would be effective for the treatment of angiogenic diseases such as cancers, rheumatoid arthritis and diabetic retinopathy
 less than Formula 1 greater than 
Wherein,
R1 is a hydrogen, alkyl or allylalkyl group; R2 and R3, which may be the same or different, each is hydrogen or an alkyl group.
Angiogenesis is a complex process in which capillary blood vessels grow in a complex physiological processes (J. Folkman and M. Klagsbrun et al., Science, Vol. 235, pp 442-447, 1987; J. Folkman and Y. Shing, J. et al., Biol. Chem., Vol. 267, pp. 10931-10934, 1992). Angiogenesis is driven by a complex array of, soluble mediators, matrix molecules and accessory cells that function to fine-tune and coordinate the response in both time and space. The initiation of angiogenesis is mediated by multiple molecules that are released from a number of sources including inflammatory cells, such as mast cells and macrophage as well as a variety of tumor cells. These molecules activate the normally quiescent vascular endothelium by binding to their respective receptors. These activated endothelial cells have a characteristic set of traits which include increased cellular proliferation, elevated expression of cell adhesion molecules, increased secretion of proteolytic enzymes, increased cellular migration and invasion, and differentiation to capillary tube. These complex cellular processes should be successfully accomplished to complete angiogenesis.
Angiogenesis is plays important role in a variety of normal physiological events, including trophoblast implantation, wound healing and embryonic development. Uncontrolled angiogenesis, however, can contribute to a number of pathological processes such as rheumatoid arthritis, diabetic retinopathy, and tumor growth and metastasis.
Malignancies are characterized by the growth and spread of tumors. One crucial factor is angiogenesis. Once a tumor has appeared, every increase in tumor cell population must be preceded by an increase in new capillaries that converge on the tumor and supply the cells with oxygen and nutrients (J. Folkman, Perspect. in Biol. and Med., Vol 29, p. 10-36, 1985; N. Weidner, et al., Amer. J. Pathol., Vol. 143, pp. 401-409, 1993). Tumors may thus remain harmless and confined to their tissue of origin, as long as angiogenesis is prevented from being activated. Therefore inhibition of tumor-associated angiogenesis is a most promising approach in cancer therapy (M. S. O""Reilly, et al., Cell, Vol. 79, pp. 316-328, 1994).
Many experimental evidence supports the hypothesis that tumor angiogenesis is fundamental for the growth and metastasis of solid tumors (M. S. O""Reilly, et al., ibid. 1994; N. Weidner, et al., N. Eng. J. Med., Vol. 324, pp. 1-8, 1991). Indeed, the majority of solid tumors are not even clinically detectable until after the occurrence of neovascularization, whose induction in solid tumors is mediated by one or more angiogenic factors (J. Folkman and Y. Shing, J. Biol. Chem., Vol. 267, pp. 10931-10934, 1992). Furthermore, angiogenesis is also important in a number of other pathological processes, including arthritis, psoriasis, diabetic retinopathy, and chronic inflammation (J. Folkman, Nature Medicine, Vol 1, p. 27-31, 1995; J. W. Miller, et al., J. Pathol., Vol. 145, pp. 574-584, 1994; A. P. Adamid, et al., Amer. J. Ophthal., Vol. 118, pp. 445-450, 1994; K. Takahashi, at al., J. Clin. Invest., Vol.93, pp.2357-2364, 1994; D. J. Peacock, et al., J. Exp. Med., Vol. 175, pp. 1135-1138, 1992; B. J. Nickoloff, et al., Amer. J. Pathol., Vol. 44, pp. 820-828, 1994). Thus, clearly methods of blocking the mechanism of angiogenesis are necessary to treat, so called, angiogenic diseases.
In vitro angiogenesis assays are important for identification of potential angiogenic inhibitors and rapid screening for pharmacological inhibitors. As noted above, differentiation of endothelial cells to capillary-like structure on Matrigel is essential processes for the mechanism of angiogenesis. When endothelial cells are plated on Matrigel, a reconstituted basement membrane protein derived from the Engelbreth-Holm-Swarm mouse tumor, the cells stop proliferating and display high motility and cell-cell communication. Moreover, the cells align and form tubes proposed as models of endothelial cell differentiation, the final step of angiogenesis. These tubes are morphologically similar to capillaries in that the cells that form these tubes are polarized and a central lumen is observed. This in vitro angiogenesis assay is very useful method to evaluate antiangiogenic activity of various compounds.
In order to treat angiogenesis-related diseases, several inhibitors of the above mechanism of angiogenesis are being studied, including platelet factor 4, the fumagillin derivative AGM 1470, Interferon-alpha, thrombospondin, angiostatic steroids, and angiostatin (J. Folkman, et al., ibid., 1995; M. S. O""Reilly, et al., ibid., 1994; V. Castle, et al., J. Clin. Invest., Vol. 87, pp.1183-1888; D. Ingber, et al., Nature, Vol. 348, pp. 555-557). All of these compounds have disadvantages. For example, endostatin and angiostatin are proteins, so that they have all of the disadvantages of proteins, including the requirement for being administered parenterally. Therefore, a non-protein inhibitor, which would selectively block the underlying mechanism of angiogenesis without adversely affecting other physiological functions, and which could be administered by many different routes, would be extremely useful. Therefore continuous development of angiogenesis inhibitors having less toxicity and more excellent effect is further required.
Based on the above, the present inventors have performed extensive screening of microbial metabolites to solve the problems described above using in vitro angiogenesis assays, and as a result, discovered 6,8-dihydroxy-4-(1-hydroxyethyl)-isocoumarin and a novel 6,8-dihydroxy-4-acetyl-isocoumarin from a fungal strain of soil. These, two isocoumarin derivatives are highly effective in inhibiting angiogenesis in vitro and in vivo. Finally, the inventors of the present invention found these compounds and their general alkyl or allyl derivatives as therapeutics of angiogenic diseases.
The invention provides novel isocoumarin derivatives inhibiting angiogensis represented by formula 1, including a method for preparing isocoumarin derivatives.
The invention also provides pharmaceutical compositions comprising isocoumarin derivatives as pharmaceutically active ingredients.
The present invention provides novel isocoumarin derivatives inhibiting angiogensis represented by the formula 1;
 less than Formula 1 greater than 
Wherein,
R1 is a hydrogen, alkyl or allylalkyl group; R2 and R3, which may be the same or different, each is hydrogen or an alkyl group.
In addition, the present invention provides 6,8-dihydroxy-4-acetyl-isocoumarin represented by formula 2 as a preferable embodiment of angiogenesis-inhibiting isocoumarin derivatives. 
The 6,8-dihydroxy-4-acetyl-isocoumarin can be prepared from fungus, Sesquicllium sp. Y70832, of soil.
The present invention provides a method of purification the active ingredients from a culture broth of the Sesquicillium sp. Y70832 using solvent extraction, column chromatography, and high performance liquid chromatography.
Additionally, the present invention provides pharmaceutical compositions comprising as a pharmaceutically active ingredient the compound of the formula 1, especially 6,8-dihydroxy-4-acetyl-isocoumarin, which is effective for the treatment of angiogenic diseases, such as cancers, rheumatoid arthritis and diabetic retinopathy.
Also, the present invention provides pharmaceutical compositions comprising as a pharmaceutically active ingredient a compound of the following formula 3, which is effective for the medical treatment of angiogenic diseases, such as cancers, rheumatoid arthritis and diabetic retinopathy. 
The pharmaceutical compositions comprising compounds of the present invention can be administered, in combination with other anti-cancer agents for the treatment of cancer or antiinflammatory drugs for the treatment of rheumatoid arthritis and diabetic retinopathy.
Hereinafter, the present invention is described in detail 6,8-dihydroxy-4-acetyl-isocoumarin represented by the formula 2 is prepared from fungi as follows.
First, Sesquicillium sp. Y70832, which can be isolated from soil, is cultured. The culture is extracted with acetone, concentrated and then extracted again with ethyl acetate. Next, the ethyl acetate extract is purified by silica gel column chromatography using a mixed solvent of methylene chloride and methanol to afford an active fraction. Further purification can be made on the active fraction through Sephadex LH20 column chromatography. From the purified extract, two active ingredients A and B are separated by high performance liquid chromatography using a mixed solvent of acetonitrile and water.
Through instrumental analysis, the structure of active ingredients A and B were identified as 6,8-dihydroxy-4-(1-hydroxyethyl)-isocoumarin of the formula 3 and a novel 6,8-dihydroxy-4-acetyl-isocoumarin of the formula 2, respectively.
6,8-Dihydroxy-4-acetyl-isocoumarin represented by the formula 2, can be converted into the isocoumarin derivatives of the formula 1 by introducing alkyl or alkylallyl group into the carbon of position 3 and/or alkyl ether group into the carbon of positions 6 and/or 8.
In order to determine the inhibitory effect of the isocoumarin derivatives against the angiogenesis, in vitro, endothelial cells, HUVEC (human umbilical vein endothelial cell) are selected. Under a specific culture condition, endothelial cells are migrated and differentiated into capillary tubes. Similar to the angiogenesis in vivo, the tube formation assay may be a good model to determine the influence of the isocoumarin derivatives on angiogenesis.
Various concentrations of the two isocoumarin derivatives are added to endothelial cells to monitor under a microscope whether the isocoumarin derivatives inhibited the differentiation of endothelial cells into capillary tubes. From this experiment, the two isocoumarin derivatives are found to inhibit the angiogenesis in concentration-dependent manners. In detail, 6,8-dihydroxy-4-(1-hydroxyethyl)-isocoumarin shows an effective inhibitory activity against the differentiation of HUVEC into a capillary tube down to the concentration of 4 xcexcg/ml. However, the differentiation of HUVEC into a capillary tube is inhibited by 6,8-dihydroxy-4-acetyl-isocoumarin down to the concentration of as low as 0.2 xcexcg/ml. 6,8-Dihydroxy-4-acetyl-isocoumarin is thus superior in anti-angiogenesis activity to 6,8-dihydroxy-4-(1-hydroxyethyl)-isocoumarin and exhibits no cytotoxicity on HUVEC.
Chorioallantoic membrane (CAM) assay was used to measure whether the two isocoumarin derivatives are effective to inhibit angiogenesis in vivo. Fertilized chick eggs were kept in a humidified incubator at 37xc2x0 C. After 3 days incubation, egg albumin were removed with hypodermic needle to allow CAM and yolk sac to drop away from the shell membrane. On day 3.5, the shell was punched out and removed to be a circular window and the shell membrane was removed. For testing of angiogenesis inhibition, a pieces of thermanox coverslip was coated with various concentration of the isocoumarin derivatives. The thermanox coverslip was placed on the chorioallantoic membranes at 4.5-day old chick embryo. After 2 days incubation at 37xc2x0 C., 10% fat emulsion was injected into chorioallantois and observed blood vessel formation during an embryogenesis or development procedure. As a positive control, retinoic acid was used and non-treated coverslip for negative control. When the CAM showed avascular zone to similar degree of retinoic acid treated CAM that had little vessels compared to negative control, the response was scored as positive, and calculated by the percentage of positive eggs to total numbers of tested eggs. 6,8-Dihydroxy-4-(1-hydroxyethyl)-isocoumarin inhibited angiogenesis by 27.3% at a concentration of 1 xcexcg/egg and by 83.3% at concentration of 10 xcexcg/egg. On the other hand, 6,8-dihydroxy-4-acetyl-isocoumarin inhibited angiogenesis by 50.0% at a concentration of 1 xcexcg/egg and by 93.3% at concentration of 10 xcexcg/egg.
From this result, it was found that 6,8-dihydroxy-4-acetyl-isocoumarin is far superior to 6,8-dihydroxy-4-(1-hydroxyethyl)-isocoumarin in inhibition of angiogenesis.
6,8-Dihydroxy-4-(1-hydroxyethyl)-isocoumarin known by the common name sescandelin, was reported to facilitate generation of root (Y. Kimura et al. , Agric. Biol. Chem., 54, 2477-2479, 1990), but has not yet been reported as to its inhibitory effect of angiogenesis. In the present invention, 6,8-dihydroxy-4-(1-hydroxyethyl)-isocoumarin is for the first time found to be useful in inhibiting angiogenesis.
Pharmaceutical compositions comprising 6,8-dihydroxy-4-acetyl-isocoumarin of the present invention as a pharmaceutically active ingredient, are useful for inhibiting proliferation, infiltration and metastasis of cancers as well as other angiogenic diseases, such as rheumatoid arthritis, diabetic retinopathy, etc., through its inhibition of angiogenesis.
In addition, a pharmaceutical compositions comprising 6,8-dihydroxy-4-(1-hydroxyethyl)-isocoumarin of the present invention as a pharmaceutically active ingredient, are also useful for inhibiting proliferation, infiltration and metastasis of cancers as well as other angiogenic diseases, such as rheumatoid arthritis, diabetic retinopathy, etc., through its inhibition of angiogenesis.
The compounds of the present invention may be administered via an oral and a parental route with general formulation. They may be used in combination with a variety of other anti-cancer agents, such as doxorubicin, taxol, etopoxide, camptothecin, 5-fluorouracil, methotrexate, or platinum complex compounds to reduce the toxicity of these anti-cancer agents and to potentiate the therapeutic efficiency. These compounds may be also administered in combination with antiinflammatory drugs for rheumatoid arthritis or drugs for diabetic retinopathy.
As mentioned above, the compounds of the present invention may be administered via oral or parental routes with various formulation. They can be formulated along with pharmaceutically acceptable diluents or expedients, such as generally-used fillers, extenders, binders, wetting agents, disintegrating agents, surfactant, etc. Solid formulation for oral administration is tablets, pills, dusting powder, granules and capsules. This solid formulations are prepared by mixing with more than one excipient, for example starches, calcium carbonate, sucrose, lactose and gelatine. Also, lubricants, such as magnesium stearate is used besides simple excipients. Liquid formulations for oral administration are suspensions, solutions, emulsions and syrups. The above-mentioned formulations can contain various excipients, such as wetting agents, sweeteners, aromatics and preservatives in addition to generally-used simple diluents, such as water and liquid paraffin. Formulations for perenteral administration are sterilized. aqueous solutions, water-insoluble excipients, suspensions, emulsions, freezing-drier and suppositories. Water-insoluble excipients and emulsions can contain propylene glycol, polyethylene glycols, vegetable oil, such as olive oil and injectable ester such as ethyoleate. Suppositories can contain witepsol, microgoal, tween 61, cacao fat, laurine fat and glycerolgelatin.
The dosage units can contain, for example, 1, 2, 3 or 4 individual doses or xc2xd, ⅓ or xc2xc of an individual dose. An individual dose preferably contains the amount of active compound which is administered in one application and which usually corresponds to a whole, one half, one third or a quarter of a daily dose.
Effective amount of the present invention is about 1 to 50, preferably 5 to 20 mg/kg of body weight and 1 to 3 times a day can be administered.